The apoptosis induction effect of Oligochitosan on
human hepatocyte and hepatoma cells
Abstract. Chitosan oligosaccharide (cso), the degradation products of chitin, are
confirmed having a wide range of physiological functions and biological activity. In this study, we explored the apoptosis induction effect of COS on liver cancer cells. Our data indicated that with 0.5-2.5 mg/ml oligochitosan effect 24 h, there was a dose-dependent effect, and the inhibition of hepatoma cells was significantly greater than the liver cells. On hepatoma cells and liver cells the IC50 was 2.5 mg/ml, and 3.0-4.0 mg/ml. Treated with 2.5 mg/ml COS for 24 h, the apoptosis of hepatoma cells increased to 76.04%, and the Bcl-2 expression of hepatoma cells reduced, while Caspase-3 increased, but it did not affect the two proteins of hepatocytes. Our results will provide an experimental basis for the clinical development of new toxic side effects of anti-liver cancer drug.
Keywords: Oligochitosan, Chang Liver, SMMC-7721, Apoptosis.
Introduction
Hepatocellular carcinoma is a common malignancy and its incidence is rising recently in the developing world. More than 700,000 new cases are diagnosed throughout the world and more than 600,000 deaths are due to hepatocellular carcinoma each year (1). Hepatocellular carcinoma becomes the seventh most common cancer worldwide, and the third leading cause of cancer-related deaths (2). Convertional chemotherapy is not efficient for liver cancer patients, because of drug resistance the toxic side effects (3). Therefore it is urgent to find a novel therapeutic drug.
Oligochitosan, obtained by hydrolysis or degradation of chitin (3), has 3-10 saccharide (N-acetyl-glucosamine or glucosamine) residues (4). Study has been indicated that Oligochitosan has a wide range of biological activities. In particular, Oligochitosan have been reported to have the health benefits such as immunity regulation, anti-tumor, anti-diabetic, antioxidant and liver protection (5,6). However, the apoptosis induction effect of Oligochitosan on tumor cells was rarely reported.
The aim of this study was to explore the apoptosis effect of Oligochitosan on human hepatocyte and hepatoma cells, which provided an experimental basis for the clinical development of novel anti-liver cancer drug.
Materials and Methods
Cell culture. The Chang Liver cells were obtained from Biochemisty department of Dalian Medical University and SMMC-7721 cells were obtained from ATCC. Both cell lines were cultured in RPML-1640 medium supplemented with 10% fetal calf serum and antibiotics at 37℃, in a humidified incubator with 5% CO2 (1).
MTT assays. The cells were plated in 96-cell plates at a density of 4×104/well, and 20 μl medium containing 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 mg/ml COS were added into each well. 20 μl PBS and 5-Fu (0.5mg/ml) were added as the negative control and the positive control. Each concentration of COS was repeated in six wells, then cultured for 24 h and 48 h and incubated with 20 μl MTT solution (5 mg/ml) for 4 h. The cells were lysed in 150 μl DMSO, and the absorbance at 589 nm was determined with a Multiskan Ascent plate reader (1).
Hoechst 33258 fluorescent staining to detect apoptosis. The cells (1.5×105/well) were seeded into 6-well plate. After 24 h incubation, COS solution (2.5 mg/ml) was added into the plate. After 24 h drug treatment, the cells were fixed in 1 ml of 4% (V/V) formaldehyde at 4℃ for 10 min. Then 1 ml staining solution of Hoechst 33258 was added into each well and keep in dark for 10 min at room temperature. The cells were observed at the wavelength of 340 nm by a fluorescence microscopy.
Cell-cycle analysis and Annexin-V/PI staining assay. The cells (1.0×106/ml) were harvested by 0.25% trypsin solution and inoculated at 25 cm2 cell culture flask. Chang Liver and SMMC-7721 cells seeded into six well plates. After 24 h drug treatment,
the cells were harvested and washed by PBS at 4℃. With 5μl Annexin-V/PI double staining buffer resuspended cells. The cells were stained with Annexin-V and PI solution for 10 min in dark and analyzed by flow cytometry.
Western blot analysis. The cells were harvested and solubilized in cold RIPA buffer. Proteins were separated by SDS-PAGE and transferred to nitrocellulose (NC) membranes by semi-dry apparatus for 40 min and then blocked with blocking buffer for 1 h. The specific primary antibody 1:500 at optimized dilution was added and incubated overnight at 4℃. After incubation with secondary antibody 1:3000 for 2 h, protein bands were visualized by ECL kit.
Statistical analysis. Data were expressed as the mean ± SD. Data are expressed as the mean ± SD. All statistical analyses were performed with standard statistical programs SPSS 11.0. A one?way ANOVA was used for the statistical analysis. P<0.05 was considered to indicate a statistically significant difference.
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